A beta-carrageenan and beta-cellobiose bifunctional enzyme and application thereof

Abstract

The application belongs to the technical field of bioengineering and its application, and particularly relates to a beta-gracilaria dura and beta-agarose bifunctional enzyme and application thereof. The amino acid sequence is shown as SEQ ID NO. 1, the beta-gracilaria dura and the beta-agarose can be degraded by the endo-acting mode, and different molecular weight oligosaccharides are generated. The application can be applied to the fields of food industry, chemical analysis and the like, and can also be applied to preparation of different molecular weight beta-gracilaria dura, beta-agarose oligosaccharides and fine analysis of the complex structure of the beta-gracilaria dura.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and its application technology, specifically relating to a bifunctional enzyme of β-heliotropium and β-agarose and its application. Background Technology

[0002] Seaweed gum and agarose are both important galactan polysaccharides derived from red algae. Seaweed gum is mainly found in the cell walls and intercellular spaces of *Gastrodia* species of red algae, and its structure consists of alternating (1-3)-O-β-D-galactopyranose-6-sulfate residues (G6S residues) and (1-4)-O-3,6-endoether-α-L-galactopyranose residues (LA residues). However, like many natural polysaccharides, seaweed gum is not completely homogeneous in structure, exhibiting significant heterogeneity. This structural complexity makes natural seaweed gum a microscopically heterogeneous molecular population, which also increases the complexity of its structural analysis. Despite its structural heterogeneity, seaweed gum still possesses good thickening, adhesive, and gelling properties, and is widely used in the textile, building materials, and coating industries. Furthermore, studies have confirmed that seaweed gum and its degradation products have various physiological activities, including anti-inflammatory, antitumor, and antiviral effects.

[0003] Agarose, a neutral polysaccharide extracted from red algae such as Gracilaria and Agaricus, is the main component of commercial agar. Its structural unit consists of alternating links of (1-3)-O-β-D-galactopyranose (G residues) and (1-4)-O-3,6-endoether-α-L-galactopyranose (LA residues). Due to its unique temperature-reversible gelation properties, high gel strength, and biocompatibility, agarose is widely used in the food industry, microbiology, and biotechnology. Studies have shown that oligosaccharides obtained from the degradation of agarose also possess various physiological functions, including antioxidant and antibacterial properties.

[0004] However, polysaccharides generally suffer from problems such as large molecular weight, high viscosity, poor solubility, and low bioavailability, which limit their direct application. Degrading polysaccharides to obtain low molecular weight seaweed gum and agarose can significantly improve their water solubility and bioavailability, making them potential functional food ingredients with broad prospects in the food and pharmaceutical fields. Enzymatic degradation aligns with the trend of green and clean production. Glycoside hydrolases can specifically cleave glycosidic bonds without destroying the natural substituents of polysaccharides. Compared with physical or chemical methods, enzymatic methods offer milder reaction conditions, higher specificity, controllable processes, and environmental friendliness, while also better preserving the original structure of the polysaccharide and exhibiting high reproducibility.

[0005] The complexity of polysaccharide structures places high demands on their degrading enzymes. Polysaccharide hydrolases typically act only on specific types of glycosidic bonds or have a strict preference for sulfation modes, leading to low degradation efficiency or a single product. For example, Chinese patent CN110951803A discloses a method and application for preparing high-purity neo-agarbiose using a combination of specific agarases, involving a bifunctional enzyme AgaB; AgaB is a bifunctional exonuclease that simultaneously possesses the activity of degrading agarose and Porphyra polysaccharide (i.e., sulfated agarose), and can degrade agar or crude agar (red algae polysaccharide extract) or its oligosaccharides to produce a single product, neo-agarbiose, the amino acid sequence of which is shown in SEQ ID NO.2. Chinese patent CN118048370A discloses a Porphyra carotene hydroxylase gene and its encoded protein and applications, wherein the protein encoded by the carotene hydroxylase gene is characterized as an ancient bifunctional enzyme derived from red algae. However, a bifunctional β-glucanase / β-agarose enzyme that can act on both glucan and agarose substrates has not yet been reported. Summary of the Invention

[0006] The technical problem to be solved by this invention is the lack of bifunctional enzymes that can simultaneously and efficiently degrade seaweed gum and agarose. There are few enzymes available for the preparation of low molecular weight seaweed gum and agarose, and key enzymes are lacking for the fine structure analysis of seaweed gum.

[0007] To address the aforementioned issues, this invention is based on a gene discovered in the strain *Wenyingzhuangia fucanilytica*, whose original nucleotides encode 381 amino acids. Its amino acid sequence is shown in SEQ ID NO.1, and its theoretical molecular weight was predicted to be 42.72 kDa using ExPASy software. From the perspective of protein crystal structure and structural biology, this study elucidates that this enzyme simultaneously possesses hydrolytic activity for both β-gum and β-agarose, thus discovering for the first time a bifunctional enzyme for β-gum and β-agarose. Its multi-catalytic activity facilitates a more thorough deconstruction of complex polysaccharide networks, improving degradation efficiency and producing structurally diverse oligosaccharide products. Based on this, the application of the aforementioned endonuclease in the enzymatic hydrolysis of β-gum and agarose is provided, thereby overcoming key bottlenecks in the efficient acquisition and practical application of this type of enzyme, the large-scale preparation of low molecular weight β-gum, agarose, and oligosaccharides, and the fine structural analysis of β-gum.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solution: a β-gum and β-agarose bifunctional enzyme, the amino acid sequence of which is SEQ ID NO. 1, as well as an enzyme derived from SEQ ID NO. 1 that has been substituted, deleted or added to one or more amino acids and has the enzyme activity of SEQ ID NO. 1, and other sequences that have greater than 80% homology with SEQ ID NO. 1.

[0009] SEQ ID NO. 1:

[0010] MNQIKLGIVFMCLSLFSCGQTNHVSDIEDSDDIIKDDDEVTQDDGLDKEGMPSAAFSACSPKNNILYDSPVNNNVSAVNRDNYGMGSWQLVDALSDEFDYPTGETATDFTSKWKFGFVNSYTGPVPTVWTGDQVSFETINGTNRALVLEAAETGSGASRRLKCGMITSIAKSSYPLFQEAKVKISNSQLAN AVWMLSDDPGTTEEIDNVEAYGPKVRPDGTSCDFPYYADRIHLSHTFKNDGGQRLDYQPHQQTWMSRKKTTGDCSRDNEVVWSEDYHYFGVKWVSETRLEYFVDGKRVKVVDGLRVDDGIDPHSYTSCGDGLTREMHMIISHAAQTWRYPSVDAFWNSSDIKTGEHTKMRVDWIRVYSPDGNVNTRSCN.

[0011] The enzyme can act on both seaweed gum and agarose substrates simultaneously, exhibiting bifunctional catalytic activity. Its mechanism of action on seaweed gum is endo-cleavage, primarily cleaving β-1,4 glycosidic bonds to generate characteristic seaweed gum oligosaccharides and heterogeneous oligosaccharides containing methyl groups. Its mechanism of action on agarose is also endo-cleavage, primarily cleaving β-1,4 glycosidic bonds to generate characteristic agarose saturated oligosaccharides.

[0012] The nucleotide sequence corresponding to the gene encoding the above-mentioned β-gum and β-agarose bifunctional enzymes SEQ ID NO. 1 is shown in SEQ ID NO. 2, as well as all genes that can be translated from SEQ ID NO. 1.

[0013] SEQ ID NO. 2:

[0014]

[0015] This invention provides a method for preparing the above-mentioned β-gum and β-agarose bifunctional enzyme. The enzyme is heterologously expressed in systems such as Escherichia coli, Bacillus subtilis, and Pichia pastoris. The β-gum and β-agarose bifunctional enzyme can be prepared in large quantities by inducing enzyme production.

[0016] The above-mentioned bifunctional enzymes of β-hede gum and β-agarose were applied in the targeted preparation of hede gum or agarose oligosaccharides with specific structures. Oligosaccharides with different degrees of polymerization were prepared by controlling the amount of enzyme added, reaction time, or substrate concentration.

[0017] The application of the aforementioned bifunctional enzymes, β-gum and β-agarose, in the analysis of the fine structure of β-gum. The bifunctional enzymes were used to enzymatically hydrolyze β-gum, and the composition and structure of the hydrolysate were analyzed to accurately deduce the structural characteristics of β-gum.

[0018] The beneficial effects of this invention are as follows:

[0019] (1) The β-hede gum and β-agarose bifunctional enzyme gene of the present invention can be used to achieve efficient enzyme preparation by cloning and expression;

[0020] (2) The β-gum and β-agarose bifunctional enzyme of the present invention can degrade gum and agarose by endocleavage;

[0021] (3) The β-gum and β-agarose bifunctional enzyme of the present invention can realize the preparation of gum oligosaccharides and agarose oligosaccharides with different degrees of polymerization;

[0022] (4) The β-seaweed gum and β-agarose bifunctional enzyme of the present invention can achieve fine analysis of the complex structure of seaweed gum. Attached Figure Description

[0023] Figure 1 : SDS-PAGE results of the β-hede gum and β-agarose bifunctional enzyme of the present invention; the left band is the purified target protein, and the right band is the protein molecular weight standard;

[0024] Figure 2 Schematic diagram illustrating the mechanism of action of the β-seaweed gum and β-agarose bifunctional enzymes of the present invention. In 2A: action on seaweed gum; In 2B: action on agarose;

[0025] Figure 3 This diagram illustrates the production of β-gum and β-agarose bifunctional enzymes of the present invention at different molecular weights when the enzyme dosage is controlled. In diagram 3A: the enzyme acts on β-gum; in diagram 3B: the enzyme acts on agarose.

[0026] Figure 4This diagram illustrates the production of β-gum and β-agarose of different molecular weights by the bifunctional enzymes of β-gum and β-agarose under controlled reaction time conditions. In diagram 4, A represents the action on β-gum; in diagram 4, B represents the action on agarose.

[0027] Figure 5 The extraction ion current chromatogram of the β-gum and β-agarose bifunctional enzyme degradation of β-gum and agarose in this invention. In 5A: action on β-gum; in 5B: action on agarose. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, all raw materials used in the following embodiments were purchased from the market.

[0029] Example 1: Heterologous expression of β-hede gum and β-agarose bifunctional enzymes in Escherichia coli:

[0030] *E. coli* carrying the target gene for the β-hede gum and β-agarose bifunctional enzyme (as shown in SEQ ID NO. 2) was passaged in LB liquid medium containing kanamycin and cultured at 37°C and 170 rpm until the OD600 reached approximately 0.4. Isopropyl thiogalactoside was then added to induce expression at 17°C for 16 h. The bacterial cells were collected by centrifugation, resuspended in 20 mM disodium hydrogen phosphate-sodium dihydrogen phosphate (PBS) buffer, and then sonicated in an ice-water bath. The supernatant was collected by centrifugation at 4°C to obtain the crude enzyme solution containing the β-hede gum and β-agarose bifunctional enzyme. The target protein was purified using an affinity purification strategy to obtain the purified β-hede gum and β-agarose bifunctional enzyme protein. Figure 1 Analysis showed that the purified protein was a single band and the apparent molecular weight (Mw) was consistent with the theoretical Mw, indicating that the target protein was successfully cloned and expressed.

[0031] Example 2: Validation of the activity of bifunctional enzymes of β-hede gum and β-agarose:

[0032] After thoroughly mixing 50 μL of appropriately diluted enzyme solution with 50 μL of 2 mg / mL seaweed gum solution, the mixture was reacted at 35 °C for 10 min and then inactivated at 100 °C for 5 min. The inactivated enzyme solution group served as the control group under the same conditions. The reducing sugar content in the experimental and control systems was detected using the pHBH method (reducing sugar increment method), and the enzyme activity of the β-seaweed gum and β-agarose bifunctional enzymes against seaweed gum was calculated. Similarly, after thoroughly mixing 50 μL of appropriately diluted enzyme solution with 50 μL of 2 mg / mL agarose solution, the mixture was reacted at 35 °C for 10 min and then inactivated at 100 °C for 5 min. The inactivated enzyme solution group served as the control group under the same conditions. The reducing sugar content in the experimental and control systems was detected using the pHBH method (reducing sugar increment method), and the enzyme activity of the β-seaweed gum and β-agarose bifunctional enzymes against agarose was calculated. 1 U of activity was defined as the activity required to generate 1 μmol of reducing sugar within 1 min. The activity of β-glucanase to β-glucan and β-agarose to β-agarose in 1 mL of fermentation broth was 143.61 U and 122.56 U, respectively, as determined by the pHBH method. The bifunctional β-glucanase and β-agarose enzymes of this invention can be successfully expressed in an *E. coli* system and can be used for the production of chemical reagents.

[0033] Example 3: HPLC analysis of the reaction process of β-heliotropium and β-agarose bifunctional enzymes:

[0034] 1 U of the enzyme solution obtained in Example 1 was added to a 100 mg / mL solution of seaweed gum and agarose, and pH 7.0 PBS buffer was added to bring the reaction volume to 100 mL. The reaction was carried out at 35 °C, and samples were taken at different times (10 min, 30 min, 1 h, 2 h, 4 h, 12 h, 24 h). The reaction was terminated by inactivation at 100 °C for 10 min. The enzymatic hydrolysis products were analyzed by HPLC. HPLC analysis conditions: column: Superdex 30 increase 3.2 / 300 GL; mobile phase: 20% acetonitrile containing 10 mM ammonium formate; flow rate: 0.075 mL / min. The results are as follows: Figure 2 As shown, in the initial stage of the reaction (within 10 min), the position of the high molecular weight exclusion peak shifted significantly backward and the signal intensity weakened, while oligosaccharide products with different degrees of polymerization were detected. As the enzymatic hydrolysis continued, the high molecular weight oligosaccharides in the system showed a trend of first accumulating and then degrading, while the low molecular weight oligosaccharides continued to accumulate and increase, indicating that the enzyme acts on the seaweed gum substrate and agarose substrate in an endo-cleavage manner.

[0035] Example 4: Different molecular weights of seaweed gum and agarose can be prepared by controlling the amount of enzyme added:

[0036] The recombinant enzyme obtained from the *E. coli* system in Example 1 was added to the seaweed gum solution and agarose solution, and the reaction was carried out at a ratio of 1 g substrate (0.5 mg / mL) corresponding to 1 U, 2 U, 4 U, 6 U, 8 U, and 10 U of recombinant enzyme. After reacting at 35°C for 1 h, 500 μL of each enzyme was inactivated. The molecular weight of seaweed gum and agarose was monitored using high performance size exclusion chromatography-differential refractive index detection-multiple-angle laser light scattering (HPSEC-MALLS-RI method). The mobile phase was 0.15 M NaCl containing 10 mM PBS at pH 7.4, and the flow rate was 0.5 mL / min. The molecular weight determination results are as follows: Figure 3 As shown, with the increase of enzyme dosage, seaweed gum was degraded to obtain low molecular weight polysaccharides of 1 kDa-100 kDa within 1 h, and agarose was degraded to obtain low molecular weight polysaccharides of 1 kDa-50 kDa.

[0037] Example 5: Different molecular weights of seaweed gum and agarose can be prepared by controlling the reaction time:

[0038] The recombinant enzyme obtained from the *E. coli* system in Example 1 was added to the seaweed gum solution and agarose solution, with a reaction ratio of 1 g substrate (0.5 mg / mL) corresponding to 1 U of recombinant enzyme. After reacting at 30°C for 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min, 500 μL of each enzyme was taken for inactivation. The molecular weights of the seaweed gum and agarose were monitored using the HPSEC-MALLS-RI method, with the mobile phase conditions the same as in Example 4. The molecular weight detection results are as follows: Figure 4 As shown, with the extension of reaction time, seaweed gum can be degraded into low molecular weight polysaccharides of 1 kDa-120 kDa within 1 h, and agarose can be degraded into low molecular weight polysaccharides of 1 kDa-50 kDa.

[0039] Example 6: LC-MS analysis of the final products of β-hede gum and β-agarose bifunctional enzyme degradation of hede gum:

[0040] 1 U of the enzyme solution obtained in Example 1 was added to a 100 mg / mL seaweed gum solution, and pH 7.0 PBS buffer was added to bring the reaction volume to 100 mL. The reaction was carried out at 35°C for 2 h, and then terminated by inactivation at 100°C for 10 min. The enzyme digest was analyzed by LC-MS using a single-pole mass spectrometer. Figure 5 As shown, mass spectrometry analysis revealed that the degradation products of seaweed gum contained typical oligosaccharides (disaccharides, tetrasaccharides, and hexasaccharides) as well as heterogeneous oligosaccharides containing methyl groups (methylated disaccharides, methylated tetrasaccharides, and dimethylated tetrasaccharides), indicating that the enzyme has the ability to degrade both the typical and heterogeneous structures of seaweed gum.

[0041] Example 7: One-dimensional and two-dimensional NMR analysis of the degradation products of β-hede gum and β-agarose by bifunctional enzymes:

[0042] 1 U of the enzyme solution obtained in Example 1 was added to a 100 mg / mL seaweed gum solution, and pH 7.0 PBS buffer was added to bring the reaction volume to 100 mL. The reaction was carried out at 35°C for 2 h, and then terminated by inactivation at 100°C for 10 min to obtain oligosaccharide samples. The lyophilized oligosaccharide samples were exchanged with heavy water twice, then dissolved again with heavy water (200 μL) and transferred to micro NMR tubes. Determination 1 H NMR and two-dimensional 1 H- 1 H-correlation spectra (COSY, TOCSY, and NOESY). Nuclear magnetic resonance spectroscopy was performed using a Bruker 600 MHz AVANCE III spectrometer at 25°C, with sodium 2,2-dimethyl-2-silylpentane-5-sulfonate as the external standard. Based on the NMR results, the characteristic disaccharide sequence of sea lettuce was deduced to be LAα1→3G6S. As an agar-type galactan, the polysaccharide structure of sea lettuce was confirmed to consist of alternating α-1,3 and β-1,4 glycosidic bonds. Based on the action of glycoside hydrolases, it can also be confirmed that this enzyme hydrolyzes the β-1,4 glycosidic bond between G6S and LA.

[0043] Based on the results of Examples 4-7, by precisely controlling the amount of enzyme added and the reaction time, controllable enzymatic hydrolysis of low molecular weight seaweed gum and agarose can be achieved, enabling the targeted preparation of low molecular weight polysaccharide products with different molecular weight ranges. This method features mild reaction conditions, well-defined product structures, and a green and efficient process, laying a solid technical and material foundation for the development of functional foods, health products, and pharmaceutical raw materials based on low molecular weight seaweed gum and agarose.

[0044] Finally, it should be noted that although the above embodiments describe specific implementations of the present invention, they are not intended to limit the invention. Those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention is defined by the appended claims. All modifications or equivalent substitutions should be included within the scope of protection of the present invention.

Claims

1. The application of a bifunctional enzyme of β-gum and β-agarose in the targeted preparation of gum or agarose oligosaccharides with specific structures, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. A bifunctional enzyme combining β-hede gum and β-agarose is used in the determination of the fine structure of hede gum, characterized in that: The amino acid sequence is shown in SEQ ID NO. 1.

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